Accumulating evidence implicates a role for protein synthesis in the formation of long-term memories and a loss of translational control with disorders characterized by cognitive dysfunction, such as fragile X syndrome and autism. Little, however, is known about how mRNA translation is regulated at synapses. The proposed experiments will define a role for S6 phosphorylation in regulating local protein synthesis using a model system that selectively activates the perforant path projections to the hippocampus. These experiments will identify patterns of synaptic activity that are most effective and define signal transduction pathways that activate mTOR-dependent protein synthesis. Our preliminary studies reveal that unilateral high frequency stimulation of the perforant path strongly activates phosphorylation of S6 in granule cells of the dentate gyrus and in the portion of the dendrites contacted by active synapses suggesting a role for S6 phosphorylation in regulating activity-dependent protein synthesis. The central hypothesis is that S6 phosphorylation acts as a signal to initiate translation of activity-dependent proteins at activated synapses. To understand the role of S6 phosphorylation in regulating translation this project aims to: 1) determine specific patterns of activity that optimally activate phosphorylation of S6, 2) define signaling pathways involved in regulating phosphorylation of S6, and 3) determine if induction of S6 phosphorylation increases mTOR-dependent protein synthesis in cell bodies, dendrites or both. Localization of S6 phosphorylation will be assessed using immunofluorescence techniques and 2-photon laser scanning imaging. Critical signaling cascades will be defined using pharmacological techniques. Levels of protein synthesis will be assessed by pulse labeling and film autoradiography. Understanding how translation of new proteins is regulated at active synapses may provide novel insights on mechanisms involved in activity-dependent synaptic modifications and on disorders involving disruptions of synaptic protein synthesis. These studies may also serve to inform subsequent analyses where physiological stimulation may be used to activate mTOR-dependent protein synthesis in cortical motor neurons to enable regeneration of axons after spinal cord injury.